Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies, largely due to its dense fibrotic stroma that promotes drug resistance and tumor progression. While patient-derived organoids (PDOs) have emerged as promising tools for modeling PDAC and evaluating therapeutic responses, the current PDO models grown in soft matrices fail to replicate the tumor's stiff extracellular matrix (ECM), limiting their predictive value for advanced disease.

We developed a biomimetic model using gelatin-based matrices of varying stiffness, achieved through modulated transglutaminase crosslinking rates, to better simulate the desmoplastic PDAC microenvironment. Using this platform, we investigated organoid morphology, proliferation, and chemoresistance to gemcitabine (Gem) and its lipophilic derivative, 4-N-stearoyl gemcitabine (Gem-S). Mechanistic studies focused on the interplay between ECM stiffness, hypoxia-inducible factor (HIF) expression, and the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway in drug resistance.

PDAC organoids in stiffer matrices demonstrated enhanced stemness features, including rounded morphology and elevated cancer stem cell (CSC) marker expression. Matrix stiffness-induced gemcitabine resistance correlated with the upregulation of ABC transporters and oxidative stress adaptive responses. While gemcitabine activated Nrf2 expression, promoting oxidative stress mitigation, Gem-S suppressed Nrf2 levels and induced oxidative stress, leading to increased reactive oxygen species (ROS) and enhanced cell death. Both compounds reduced HIF expression, with gemcitabine showing greater efficacy.

Our study reveals ECM stiffness as a critical mediator of PDAC chemoresistance through the promotion of stemness and modulation of Nrf2 and HIF pathways. Gem-S demonstrates promise in overcoming gemcitabine resistance by disrupting Nrf2-mediated adaptive responses and inducing oxidative stress. These findings underscore the importance of biomechanically accurate tumor models and suggest that dual targeting of mechanical and oxidative stress pathways may improve PDAC treatment outcomes.

Hydrogels and microgels are emerging as pivotal platforms in biomedicine, with significant potential in targeted drug delivery, enhanced infection management, and tissue repair and regeneration. These gels, characterized by their high water content, unique structures, and adaptable mechanical properties, interact seamlessly with biological systems, making them invaluable for controlled and targeted drug release. In the realm of infection management, hydrogels and microgels can incorporate antimicrobial agents, offering robust defenses against bacterial infections. This capability is increasingly important in the fight against antibiotic resistance, providing innovative solutions for infection prevention in wound dressings, surgical implants, and medical devices. Additionally, the biocompatibility and customizable mechanical properties of these gels make them ideal scaffolds for tissue engineering, supporting the growth and repair of damaged tissues. Despite their promising applications, challenges such as ensuring long-term stability, enhancing therapeutic agent loading capacities, and scaling production must be addressed for widespread adoption. This review explores the current advancements, opportunities, and limitations of hydrogels and microgels, highlighting research and technological directions poised to revolutionize treatment strategies through personalized and regenerative approaches.

Bone morphogenetic proteins are essential for bone regeneration/fracture healing but can also induce heterotopic ossification (HO). Understanding accessory factors modulating BMP signaling would provide both a means of enhancing BMP-dependent regeneration while preventing HO. This study focuses on the ability of the collagen receptor, discoidin domain receptor 2 (DDR2), to regulate BMP activity. As will be shown, induction of bone formation by subcutaneous BMP2 implants is severely compromised in Ddr2-deficient mice. In addition, Ddr2 deficiency attenuates HO in mice expressing the ACVR1 mutation associated with human fibrodysplasia ossificans progressiva. In cells migrating into BMP2 implants, DDR2 is co-expressed with GLI1, a skeletal stem cell marker, and DDR2/GLI1-positive cells participate in BMP2-induced bone formation where they contribute to chondrogenic and osteogenic lineages. Consistent with this distribution, conditional knockout of Ddr2 in Gli1-expressing cells inhibited bone formation to the same extent seen in globally Ddr2-deficient animals. This response was explained by selective inhibition of Gli1+ cell proliferation without changes in apoptosis. The basis for this DDR2 requirement was explored further using bone marrow stromal cells. Although Ddr2 deficiency inhibited BMP2-dependent chondrocyte and osteoblast differentiation and in vivo, bone formation, early BMP responses including SMAD phosphorylation remained largely intact. Instead, Ddr2 deficiency reduced the nuclear/cytoplasmic ratio of the Hippo pathway intermediates, YAP and TAZ. This suggests that DDR2 regulates Hippo pathway-mediated responses to the collagen matrix, which subsequently affect BMP responsiveness. In summary, DDR2 is an important modulator of BMP signaling and a potential therapeutic target both for enhancing regeneration and treating HO.

Biomaterial-induced macrophage-derived multinucleated cells (MNCs) are often observed at or near material implantation sites, yet their subtypes and roles in tissue repair and wound healing remain unclear. This study compares material-induced MNCs to cytokine-induced MNCs using both in vitro and in vivo models. 3D-embedded Raw264.7 cells and rat bone marrow-derived monocytes (BMDMs), with or without cytokines such as IL-4 and RANKL, were characterized for their MNC morphologies and subtypes via in situ immunocytochemistry and flow cytometry. Macrophage polarization and osteoclastic differentiation were assessed through NO production, arginase activity, and tartrate-resistant acid phosphatase levels. 3D matrix-induced MNCs expressed the same phenotypic heterogeneity as the IL-4 and RANK-treated ones. 3D matrix-induced MNCs displayed the same phenotypic heterogeneity as those treated with IL-4 and RANKL. A high viscoelastic matrix (1006.48 ± 92.29 Pa) induced larger populations of proinflammatory and osteoclast-like MNCs, whereas a low viscoelastic matrix (38.61 ± 7.56 Pa) supported active differentiation and gene expression across pro-, anti-inflammatory, and osteoclast-like macrophages. Matrix viscoelasticity also influenced the effects of IL-4 and RANKL on macrophage-derived MNC polarization. In an in vivo subcutaneous implantation model, medium to high viscoelastic matrices exhibited higher populations of CD86+ and RANK+ MNCs, while low viscoelastic matrices showed higher populations of CD206+ MNCs. These findings suggest that matrix viscoelasticity modulates macrophage differentiation and MNC phenotype, with low viscoelastic matrices potentially favoring anti-inflammatory MNCs and macrophage differentiation suitable for subcutaneous implantation.

Delivery of therapeutic stem cells to treat bone tissue damage is a promising strategy that faces many hurdles to clinical translation. Among them is the design of a delivery vehicle which promotes desired cell behavior for new bone formation. In this work, we describe the use of an injectable microporous hydrogel, made of crosslinked gelatin microgels, for the encapsulation and delivery of human mesenchymal stem cells (MSCs) and compared it to a traditional nonporous injectable hydrogel. MSCs encapsulated in the microporous hydrogel showed rapid cell spreading with direct cell-cell connections whereas the MSCs in the nonporous hydrogel were entrapped by the surrounding polymer mesh and isolated from each other. On a per-cell basis, encapsulation in microporous hydrogel induced a 4 × increase in alkaline phosphatase (ALP) activity and calcium mineral deposition in comparison to nonporous hydrogel, as measured by ALP and calcium assays, which indicates more robust osteogenic differentiation. RNA-seq confirmed the upregulation of the genes and pathways that are associated with cell spreading and cell-cell connections, as well as the osteogenesis in the microporous hydrogel. These results demonstrate that microgel-based injectable hydrogels can be useful tools for therapeutic cell delivery for bone tissue repair.

Cell encapsulation within three-dimensional hydrogels is a promising approach to mimic tissues. However, true biomimicry of the intricate microenvironment, biophysical and biochemical gradients, and the macroscale hierarchical spatial organizations of native tissues is an unmet challenge within tissue engineering. This review provides an overview of the macromolecular chemistries that have been applied toward the design of cell-friendly hydrogels, as well as their application toward controlling biophysical and biochemical bulk and gradient properties of the microenvironment. Furthermore, biofabrication technologies provide the opportunity to simultaneously replicate macroscale features of native tissues. Biofabrication strategies are reviewed in detail with a particular focus on the compatibility of these strategies with the current macromolecular toolkit described for hydrogel design and the challenges associated with their clinical translation. This review identifies that the convergence of the ever-expanding macromolecular toolkit and technological advancements within the field of biofabrication, along with an improved biological understanding, represents a promising strategy toward the successful tissue regeneration.

The repair of bone defects using grafts is commonly employed in clinical practice. However, the risk of infection poses a significant concern. Tissue engineering scaffolds with antibacterial functionalities offer a better approach for bone tissue repair. In this work, firstly, two kinds of nanoparticles were prepared using chitosan to complex with ciprofloxacin and BMP-2, respectively. The ciprofloxacin complex nanoparticles improved the dissolution efficiency of ciprofloxacin achieving a potent antibacterial effect and cumulative release reached 95 % in 7 h. For BMP-2 complexed nanoparticles, the release time points can be programmed at 80 h, 100 h or 180 h by regulating the number of coating chitosan layers. Secondly, a functional scaffold was prepared by combining the two nanoparticles with chitosan nanofibers. The microscopic nanofiber structure of the scaffold with 27.28 m2/g specific surface area promotes cell adhesion, high porosity provides space for cell growth, and facilitates drug loading and release. The multifunctional scaffold exhibits programmed release function, and has obvious antibacterial effect at the initial stage of implantation, and releases BMP-2 to promote osteogenic differentiation of mesenchymal stem cells after the antibacterial effect ends. The scaffold is expected to be applied in clinical bone repair and graft infection prevention.

Atherosclerosis, the primary cause of cardiovascular diseases (CVDs), is characterized by phenotypic changes in fibrous proliferation, chronic inflammation and lipid accumulation mediated by vascular endothelial cells (ECs) and vascular smooth muscle cells (SMCs) which are correlated with the stiffening and ectopic remodeling of local extracellular matrix (ECM). The native residents, ECs and SMCs, are not only affected by various chemical factors including inflammatory mediators and chemokines, but also by a range of physical stimuli, such as shear stress and ECM stiffness, presented in the microenvironmental niche. Especially, ECs, as a semi-selective barrier, can sense mechanical forces, respond quickly to changes in mechanical loading and provide context-specific adaptive responses to restore homeostasis. However, blood arteries undergo stiffening and lose their elasticity with age. Reports have shown that the ECM stiffening could influence EC fate by changing the cell adhesion, spreading, proliferation, cell to cell contact, migration and even communication with SMCs. The cell behaviour changes mediated by ECM stiffening are dependent on the activation of a signaling cascade of mechanoperception and mechanotransduction. Although the substantial evidence directly indicates the importance of ECM stiffening on the native ECs, the understanding about this complex interplay is still largely limited. In this review, we systematically summarize the roles of ECM stiffening on the behaviours of endothelial cells and elucidate the underlying details in biological mechanism, aiming to provide the process of how ECs integrate ECM mechanics and the highlights for bioaffinity of tissue-specific engineered scaffolds.

Natural polymeric nanobiocomposites hold promise in repairing damaged bone tissue in tissue engineering. These materials create an extracellular matrix (ECM)-like microenvironment that induces stem cell differentiation. In this study, we investigated a new cytocompatible nanobiocomposite made from cotton cellulose nanofibers (CNFs) combined with chitosan polymer to induce osteogenic stem cell differentiation. First, we characterized the chemical composition, nanotopography, swelling properties, and mechanical properties of the cotton CNF/chitosan nanobiocomposite scaffold. Then, we examined the biological characteristics of the nanocomposites to evaluate their cytocompatibility and osteogenic differentiation potential using human mesenchymal stem cells derived from exfoliated deciduous teeth. The results showed that the nanobiocomposite exhibited favorable cytocompatibility and promoted osteogenic differentiation of cells without the need for chemical inducers, as demonstrated by the increase in alkaline phosphatase activity and ECM mineralization. Therefore, the cotton CNF/chitosan nanobiocomposite scaffold holds great promise for bone tissue engineering applications.

Mechanics, as a key physical factor which affects cell function and tissue regeneration, is attracting the attention of researchers in the fields of biomaterials, biomechanics, and tissue engineering. The macroscopic mechanical properties of tissue engineering scaffolds have been studied and optimized based on different applications. However, the mechanical properties of the overall scaffold materials are not enough to reveal the mechanical mechanism of the cell-matrix interaction. Hence, the mechanical detection of cell mechanics and cellular-scale microenvironments has become crucial for unraveling the mechanisms which underly cell activities and which are affected by physical factors. This review mainly focuses on the advanced technologies and applications of cell-scale mechanical detection. It summarizes the techniques used in micromechanical performance analysis, including atomic force microscope (AFM), optical tweezer (OT), magnetic tweezer (MT), and traction force microscope (TFM), and analyzes their testing mechanisms. In addition, the application of mechanical testing techniques to cell mechanics and tissue engineering scaffolds, such as hydrogels and porous scaffolds, is summarized and discussed. Finally, it highlights the challenges and prospects of this field. This review is believed to provide valuable insights into micromechanics in tissue engineering.

Ossification of the posterior longitudinal ligaments (OPLL) is common disorder characterized by heterotopic ossification of the spinal ligaments. Mechanical stimulation (MS) plays an important role in OPLL. DLX5 is an essential transcription factor required for osteoblast differentiation. However, the role of DLX5 during in OPLL is unclear. This study aims to investigate whether DLX5 is associated with OPLL progression under MS.

Stretch stimulation was applied to spinal ligaments cells derived from OPLL (OPLL cells) and non-OPLL (non-OPLL cells) patients. Expression of DLX5 and osteogenesis-related genes were determined by quantitative real-time polymerase chain reaction and Western blot. The osteogenic differentiation ability of the cells was measured using alkaline phosphatase (ALP) staining and alizarin red staining. The protein expression of DLX5 in the tissues and the nuclear translocation of NOTCH intracellular domain (NICD) was examined by immunofluorescence.

Compared with non-OPLL cells, OPLL cells expressed higher levels of DLX5 in vitro and vivo (p < 0.01). Upregulated expression of DLX5 and osteogenesis-related genes (OSX, RUNX2, and OCN) were observed in OPLL cells induced with stretch stimulation and osteogenic medium, whereas there was no change in the non-OPLL cells (p < 0.01). Cytoplasmic NICD protein translocated from the cytoplasm to the nucleus inducing DLX5 under stretch stimulation, which was reduced by the NOTCH signaling inhibitors (DAPT) (p < 0.01).

These data suggest that DLX5 play a critical role in MS-induced progression of OPLL through NOTCH signaling, which provides a new insight into the pathogenesis of OPLL.

Smart biomaterials have been rapidly advancing ever since the concept of tissue engineering was proposed. Interacting with human cells, smart biomaterials can play a key role in novel tissue morphogenesis. Various aspects of biomaterials utilized in or being sought for the goal of encouraging bone regeneration, skin graft engineering, and nerve conduits are discussed in this review. Beginning with bone, this study summarizes all the available bioceramics and materials along with their properties used singly or in conjunction with each other to create scaffolds for bone tissue engineering. A quick overview of the skin-based nanocomposite biomaterials possessing antibacterial properties for wound healing is outlined along with skin regeneration therapies using infrared radiation, electrospinning, and piezoelectricity, which aid in wound healing. Furthermore, a brief overview of bioengineered artificial skin grafts made of various natural and synthetic polymers has been presented. Finally, by examining the interactions between natural and synthetic-based biomaterials and the biological environment, their strengths and drawbacks for constructing peripheral nerve conduits are highlighted. The description of the preclinical outcome of nerve regeneration in injury healed with various natural-based conduits receives special attention. The organic and synthetic worlds collide at the interface of nanomaterials and biological systems, producing a new scientific field including nanomaterial design for tissue engineering.

The well-known synergetic interplay between the skeletal and immune systems has changed the design of advanced bone tissue engineering strategies. The immune system is essential during the bone lifetime, with macrophages playing multiple roles in bone healing and biomaterial integration. If in the past, the most valuable aspect of implants was to avoid immune responses of the host, nowadays, it is well-established how important are the crosstalks between immune cells and bone-engineered niches for an efficient regenerative process to occur. For that, it is essential to recapitulate the multiphenotypic cellular environment of bone tissue when designing new approaches. Indeed, the lack of osteoimmunomodulatory knowledge may be the explanation for the poor translation of biomaterials into clinical practice. Thus, smarter hydrogels incorporating immunomodulatory bioactive factors, stem cells, and immune cells are being proposed to develop a new generation of bone tissue engineering strategies. This review highlights the power of immune cells to upgrade the development of innovative engineered strategies, mainly focusing on orthopaedic and dental applications.

Biochemical signals, such as growth factors, cytokines, and transcription factors are known to play a crucial role in regulating a variety of cellular activities as well as maintaining the normal function of different tissues and organs. If the biochemical signals are assumed to be one side of the coin, the other side comprises biophysical cues. There is growing evidence showing that biophysical signals, and in particular mechanical cues, also play an important role in different stages of human life ranging from morphogenesis during embryonic development to maturation and maintenance of tissue and organ function throughout life. In order to investigate how mechanical signals influence cell and tissue function, tremendous efforts have been devoted to fabricating various materials and devices for delivering mechanical stimuli to cells and tissues. Here, an overview of the current state of the art in the design and development of such materials and devices is provided, with a focus on their design principles, and challenges and perspectives for future research directions are highlighted.

Bioprinting is a biofabrication technology which allows efficient and large-scale manufacture of 3D cell culture systems. However, the available biomaterials for bioinks used in bioprinting are limited by their printability and biological functionality. Fabricated constructs are often homogeneous and have limited complexity in terms of current 3D cell culture systems comprising multiple cell types. Inspired by the phenomenon that hydrogels can exchange liquids under the infiltration action, infiltration-induced suspension bioprinting (IISBP), a novel printing technique based on a hyaluronic acid (HA) suspension system to modulate the properties of the printed scaffolds by infiltration action, was described in this study. HA served as a suspension system due to its shear-thinning and self-healing rheological properties, simplicity of preparation, reusability, and ease of adjustment to osmotic pressure. Changes in osmotic pressure were able to direct the swelling or shrinkage of 3D printed gelatin methacryloyl (GelMA)-based bioinks, enabling the regulation of physical properties such as fiber diameter, micromorphology, mechanical strength, and water absorption of 3D printed scaffolds. Human umbilical vein endothelial cells (HUVEC) were applied as a cell culture model and printed within cell-laden scaffolds at high resolution and cell viability with the IISBP technique. Herein, the IISBP technique had been realized as a reliable hydrogel-based bioprinting technique, which enabled facile modulation of 3D printed hydrogel scaffolds properties, being expected to meet the scaffolds requirements of a wide range of cell culture conditions to be utilized in bioprinting applications.

Human dental pulp stem cells (HDPSCs) have great potential to be used in regenerative medicine. To use these stem cells effectively for this purpose, they should be grown in a 3D cell culture that mimics their natural niches instead of a 2D conventional cell culture. The aim of this study was to grow the HDPSCs in the 3D cell culture created by Transglutaminase-crosslinked collagen hydrogels (Col-Tgel) in two different strengths to find a suitable 3D cell culture environment for these stem cells. Two stiffness of the 3D Col-Tgel were used to grow the HDPSCs: soft and medium matrix with strength of 0.9-1.5 kPa and 14-20 kPa, respectively. HDPSCs express markers similar to MSCs, therefore seven such markers were analyzed in the HDPSCs during their growth in the 2D and in the 3D soft and medium Col-Tgel. The CD105 and CD90 markers were significantly (p < 0.05) downregulated in HDPSCs cultured in both 3D cell culture conditions compared with HDPSCs in 2D cell culture. Furthermore, CD34 marker, a negative marker, expressed by a few cells in HDPSCs culture was upregulated (p < 0.05) in HDPSCs cultured in medium 3D Col-Tgel, indicating cells that expressing the marker grow better in medium 3D Col-Tgel. The apoptosis results revealed that HDPSCs in medium 3D Col-Tgel had the least number of live cells and a significantly (p < 0.05) higher early apoptosis rate compared to HDPSCs in 2D and 3D Col-Tgel medium. MTT analysis also showed a significant difference among the three cell culture conditions. We conclude that HDPSCs cultured on 3D soft Col-Tgel showed better proliferation than cells cultured in 3D medium gel. These results demonstrate that the ideal environment to grow HDPSCs in 3D is the soft Col-Tgel not medium Col-Tgel.

Three-dimensional (3D) bioprinting based on digital light processing (DLP) offers unique opportunities to prepare scaffolds that mimic the architecture and biomechanical properties of human tissues. Limited availability of biocompatible and biodegradable bioinks amenable for DLP-bioprinting is an impediment in this field. This study presents a bioink prepared from silk fibroin (SF) tailored for DLP bioprinting. Photocurable methacrylated-SF (SF-MA) was synthesized with 67.3% of methacrylation. Physical characterization of rheological and mechanical properties revealed that the 3D printed hydrogels of SF-MA (spanning from 10 to 25 wt%) exhibit bone tissue-like viscoelastic behavior and compressive modulus ranging from ≈12 kPa to ≈96 kPa. The gels exhibited favorable degradation (≈48 to 91% in 21 days). This SF-MA bioink afforded the printing of complex structures, with high precision. Pre-osteoblasts were successfully encapsulated in 3D bioprinted SF-MA hydrogels with high viability. 15% SF-MA DLP bioprinted hydrogels efficiently supported cell proliferation with favorable cell morphology and cytoskeletal organization. A progressive increase in cell-mediated calcium deposition up to 14 days confirmed the ability of the gels to drive osteogenesis, which was further augmented by soluble induction factors. This work demonstrates the potential of silk fibroin-derived bioinks for DLP-based 3D bioprinting of scaffolds for tissue engineering.

A major challenge in tissue engineering is the development of alternatives to traditional bone autografts and allografts that can regenerate critical-sized bone defects. Here we present the design of injectable pH-responsive double-crosslinked adhesive hydrogels inspired by the molecular mechanism and environmental post-processing of marine mussel adhesive. Nine adhesive hydrogel formulations were developed through the conjugation of crosslinkable catechol functional groups (DOPA) and the synthetic oligomer oligo[poly(ethylene glycol) fumarate] (OPF), varying the DOPA content (w/w%) and molecular weight (MW) of the OPF backbone to produce formulations with a range of swelling ratios, porosities, and crosslink densities. DOPA incorporation altered the surface chemistry, mechanical properties, and surface topography of hydrogels, resulting in an increase in material stiffness, slower degradation, and enhanced pre-osteoblast cell attachment and proliferation. When injected within simulated bone defects, DOPA-mediated interfacial adhesive interactions also prevented the displacement of scaffolds, an effect that was maintained even after swelling within physiological conditions. Taken together, OPF-DOPA hydrogels represent a promising new material to enhanced tissue integration and the prevention of the post-implantation migration of scaffolds that can occur due to biomechanical loading in vivo.

Mechanical cues from the extracellular matrix (ECM) microenvironment are known to be significant in modulating the fate of stem cells to guide developmental processes and maintain bodily homeostasis. Tissue engineering has provided a promising approach to the repair or regeneration of damaged tissues. Scaffolds are fundamental in cell-based regenerative therapies. Developing artificial ECM that mimics the mechanical properties of native ECM would greatly help to guide cell functions and thus promote tissue regeneration. In this review, we introduce various mechanical cues provided by the ECM including elasticity, viscoelasticity, topography, and external stimuli, and their effects on cell behaviours. Meanwhile, we discuss the underlying principles and strategies to develop natural or synthetic biomaterials with different mechanical properties for cellular modulation, and explore the mechanism by which the mechanical cues from biomaterials regulate cell function toward tissue regeneration. We also discuss the challenges in multimodal mechanical modulation of cell behaviours and the interplay between mechanical cues and other microenvironmental factors.

Alteration of the cellular microenvironment may influence the intra- and intercellular communication and contribute to cartilage injury and repair. The purpose of this study was to investigate how matrix elasticity/stiffness affects chondrogenic activities, including cell survival, phenotypic expression, and the release of both pro- and anti-inflammatory cytokines.

Human articular chondrocytes (HACs) cultured on traditional 2-dimensional (2D) plastic surfaces were compared with those cultured within 3D hydrogel matrices of varying stiffness. Chondrogenic proliferation, differentiation, and the expression of pro- and anti-inflammatory cytokines were evaluated. Both interleukin-1-beta (IL-1β) and human synovial fluid-derived cells (hSFCs) were introduced to study the effects of matrix stiffness on chondrocyte response.

Cells demonstrated the most robust chondrogenic differentiation and secreted the least pro-inflammatory cytokines when the matrix stiffness was close to their native microenvironment. The IL-1β effects were attenuated when HACs were co-cultured with hSFCs.

Modifying the matrix stiffness to mimic the native cartilage microenvironment not only optimized chondrogenic expression but also was essential for the regulation of physiological homeostasis. This study proposed a new toolkit to study cell-molecule, cell-cell, and cell-matrix influence on cartilage physiology.

Hydrogels can be fabricated and designed to exert direct control over stem cells' adhesion and differentiation. In this study, we have investigated the use of polydopamine (pDA)-treatment as a binding platform for bioactive compounds to create a versatile gelatin-alginate (Gel-Alg) hydrogel for tissue engineering applications. Precisely, pDA was used to modify the surface properties of the hydrogel and better control the adhesion and osteogenic differentiation of human adipose-derived stem cells (hASCs). pDA enabled the adsorption of different types of bioactive molecules, including a model osteoinductive drug (dexamethasone) as well as a model pro-angiogenic peptide (QK). The pDA treatment efficiently retained the drug and the peptide compared to the untreated hydrogel and proved to be effective in controlling the morphology, cell area, and osteogenic differentiation of hASCs. Overall, the findings of this study confirm the efficacy of pDA treatment as a valuable strategy to modulate the biological properties of biocompatible Gel-Alg hydrogels and further extend their value in regenerative medicine.

The apparent rise of bone disorders demands advanced treatment protocols involving tissue engineering. Here, we describe self-assembling tetrapeptide scaffolds for the growth and osteogenic differentiation of human mesenchymal stem cells (hMSCs). The rationally designed peptides are synthetic amphiphilic self-assembling peptides composed of four amino acids that are nontoxic. These tetrapeptides can quickly solidify to nanofibrous hydrogels that resemble the extracellular matrix and provide a three-dimensional (3D) environment for cells with suitable mechanical properties. Furthermore, we can easily tune the stiffness of these peptide hydrogels by just increasing the peptide concentration, thus providing a wide range of peptide hydrogels with different stiffnesses for 3D cell culture applications. Since successful bone regeneration requires both osteogenesis and vascularization, our scaffold was found to be able to promote angiogenesis of human umbilical vein endothelial cells (HUVECs) in vitro. The results presented suggest that ultrashort peptide hydrogels are promising candidates for applications in bone tissue engineering.

The aim of this study is to investigate polyacrylamide-based hydrogels stress relaxation and the subsequent impact on the osteogenic differentiation of human mesenchymal stem cells (hMSCs). Different hydrogels are synthesized by varying the amount of cross-linker and the ratio between the monomers (acrylamide and acrylic acid), and characterized by compression tests. It has been found that hydrogels containing 18% of acrylic acid exhibit an average relaxation of 70%, while pure polyacrylamide gels show an average relaxation of 15%. Subsequently, hMSCs are cultured on two different hydrogels functionalized with a mimetic peptide of the bone morphogenetic protein-2 to enable cell adhesion and favor their osteogenic differentiation. Phalloidin staining shows that for a constant stiffness of 55 kPa, a hydrogel with a low relaxation (15%) leads to star-shaped cells, which is typical of osteocytes, while a hydrogel with a high relaxation (70%) presents cells with a polygonal shape characteristic of osteoblasts. Immunofluorescence labeling of E11, strongly expressed in early osteocytes, also shows a dramatically higher expression for cells cultured on the hydrogel with low relaxation (15%). These results clearly demonstrate that, by fine-tuning hydrogels stress relaxation, hMSCs differentiation can be directed toward osteoblasts, and even osteocytes, which is particularly rare in vitro.

The demand for biomaterials that promote the repair, replacement, or restoration of hard and soft tissues continues to grow as the population ages. Traditionally, smart biomaterials have been thought as those that respond to stimuli. However, the continuous evolution of the field warrants a fresh look at the concept of smartness of biomaterials. This review presents a redefinition of the term "Smart Biomaterial" and discusses recent advances in and applications of smart biomaterials for hard tissue restoration and regeneration. To clarify the use of the term "smart biomaterials", we propose four degrees of smartness according to the level of interaction of the biomaterials with the bio-environment and the biological/cellular responses they elicit, defining these materials as inert, active, responsive, and autonomous. Then, we present an up-to-date survey of applications of smart biomaterials for hard tissues, based on the materials' responses (external and internal stimuli) and their use as immune-modulatory biomaterials. Finally, we discuss the limitations and obstacles to the translation from basic research (bench) to clinical utilization that is required for the development of clinically relevant applications of these technologies.

Extrusion-based 3D bioprinting is hampered by the inability to print materials of low-viscosity. In this study, a single initiating system based on ruthenium (Ru) and sodium persulfate (SPS) is utilized for a sequential dual-step crosslinking approach: 1) primary (partial) crosslinking in absence of light to alter the bioink's rheological profile for print fidelity, and 2) subsequent secondary post-printing crosslinking for shape maintenance. Allyl-functionalized gelatin (Gel-AGE) is used as a bioink, allowing thiol-ene click reaction between allyl moieties and thiolated crosslinkers. A systematic investigation of primary crosslinking reveals that a thiol-persulfate redox reaction facilitates thiol-ene crosslinking, mediating an increase in bioink viscosity that is controllable by tailoring the Ru/SPS, crosslinker, and/or Gel-AGE concentrations. Thereafter, subsequent photoinitiated secondary crosslinking then facilitates maximum conversion of thiol-ene bonds between AGE and thiol groups. The dual-step crosslinking method is applicable to a wide biofabrication window (3-10 wt% Gel-AGE) and is demonstrated to allow printing of low-density (3 wt%) Gel-AGE, normally exhibiting low viscosity (4 mPa s), with high shape fidelity and high cell viability (>80%) over 7 days of culture. The presented approach can therefore be used as a one-pot system for printing low-viscous bioinks without the need for multiple initiating systems, viscosity enhancers, or complex chemical modifications.

Growth factors and mechanical cues synergistically affect cellular functions, triggering a variety of signaling pathways. The molecular levels of such cooperative interactions are not fully understood. Due to its role in osteogenesis, the growth factor bone morphogenetic protein 2 (BMP-2) is of tremendous interest for bone regenerative medicine, osteoporosis therapeutics, and beyond. Here, contribution of BMP-2 signaling and extracellular mechanical cues to the osteogenic commitment of C2C12 cells is investigated. It is revealed that these two distinct pathways are integrated at the transcriptional level to provide multifactorial control of cell differentiation. The activation of osteogenic genes requires the cooperation of BMP-2 pathway-associated Smad1/5/8 heteromeric complexes and mechanosensitive YAP/TAZ translocation. It is further demonstrated that the Smad complexes remain bound onto and active on target genes, even after BMP-2 removal, suggesting that they act as a "molecular memory unit." Thus, synergistic stimulation with BMP-2 and mechanical cues drives osteogenic differentiation in a programmable fashion.

Foreign body reaction reflects the integration between biomaterials and host cells. At the implantation microenvironment, macrophages usually fuse into multinuclear cells, also known as foreign body giant cells, to respond to the biomaterial implants. To understand the biomaterial-induced macrophage fusion, we examined whether biomaterial alone can initiate and control the fusion rate without exogenous cytokines and chemicals. We introduced a collagen-based 3D matrix to embed Raw264.7 cell line and primary rat bone marrow-derived macrophages. We found the biomaterial-stimuli interacted regional macrophages and altered the overall fusogenic protein expressions to regulate the macrophage fusion rate. The fusion rate could be altered by modulating the cell-matrix and cell-cell adhesions. The fused macrophage morphologies, the nuclei number in the fused macrophage, and the fusion rates were matrix dependent. The phenomena were also observed in the in vivo models. These results suggest that the biomaterial-derived stimuli exert similar functions as cytokines to alter the competency of macrophage fusion as well as their drug sensitivity in the biomaterial implanted tissue environment. Furthermore, this in vitro 3D-matrix model has the potential to serve as a toolbox to predict the host tissue response on implanted biomaterials.

Bone tissue-derive biomaterials have become of great interest to treat diseases of the skeletal system. Biological scaffolds of demineralized and decellularized extracellular matrices (ECM) have been developed and one of these options are ECM hydrogels derived from bovine bone. Nanomaterials may be able to regulate stem cell differentiation due to their unique physical-chemical properties. The present work aimed to evaluate the osteoinductive effects of ECM hydrogels associated with barium titanate nanoparticles (BTNP) on dental pulp cells derived from exfoliated teeth. The addition of BTNP in the ECM derived hydrogel did not affect cell proliferation and the formation of bone nodules. Furthermore, it increased the expression of bone alkaline phosphatase. The results demonstrated that the nanobiocomposites were able to promote the osteogenic differentiation, even in the absence of chemical inducing factors for osteogenic differentiation. In conclusion, bovine bone ECM hydrogel combined with BTNP presented and increased expression of markers of osteogenic differentiation in the absence of chemical inducing factors.

Scaffold macroporosity has been shown to be critical for promoting bone regeneration. Although injectable materials are preferred for minimally invasive delivery, conventional macroporous scaffolds were not injectable and do not support homogeneous cell encapsulation. We recently reported a gelatin-based microribbon (μRB) scaffold that offers macroporosity while also supporting homogeneous cell encapsulation. Compared to conventional gelatin hydrogels, macroporous gelatin μRB scaffolds demonstrated great advantage in enhancing mesenchymal stem cell (MSC)-based cartilage formation. However, whether gelatin-based μRBs support MSC osteogenesis and bone formation remains unknown. The goal of this study is to assess the potential of gelatin-based μRBs for supporting MSC-based osteogenesis and bone formation in vitro. Given recent evidence from the literature that osteogenesis is sensitive to substrate stiffness, we further investigate how varying μRB stiffness modulates MSC osteogenesis. We first determine the maximal stiffness range of gelatin μRBs that can be fabricated (13-57 kPa), which supports both retention of μRB shape and macroporosity within scaffolds after inter-cross-linking. Interestingly, varying μRB stiffness across a broad range of stiffness did not significantly impact osteogenesis, with all groups supporting upregulation of bone markers and extensive collagen deposition. All gelatin μRBs also supported a comparable level of cell spreading and upregulation of mechanosensing markers. However, soft μRB (13 kPa) scaffolds did not maintain structural integrity and condensed into a pellet over time. Both intermediate and stiff gelatin μRB-based scaffolds maintained their integrity and supported robust bone formation, leading to a more than 10-fold increase in the compressive moduli of engineered bone after 5 weeks of culture in osteogenic media. Incorporating hydroxyapatite (HA) nanoparticle coating onto the gelatin μRB surface further accelerated the maturation of MSCs into osteoblasts and mineralization. Together, these results validate that gelatin μRBs can support MSC osteogenesis across a broad range of stiffness and offers an injectable macroporous scaffold for enhancing stem-cell-based bone regeneration.

In the human body, cells in a tissue are exposed to signals derived from their specific extracellular matrix (ECM), such as architectural structure, mechanical properties, and chemical composition (proteins, growth factors). Research on biomaterials in tissue engineering and regenerative medicine aims to recreate such stimuli using engineered materials to induce a specific response of cells at the interface. Although traditional biomaterials design has been mostly limited to varying individual signals, increasing interest has arisen on combining several features in recent years to improve the mimicry of extracellular matrix properties. Tremendous progress in combinatorial surface modification exploiting, for example, topographical features or variations in mechanics combined with biochemical cues has enabled the identification of their key regulatory characteristics on various cell fate decisions. Gradients especially facilitated such research by enabling the investigation of combined continuous changes of different signals. Despite unravelling important synergies for cellular responses, challenges arise in terms of fabrication and characterization of multifunctional engineered materials. This review summarizes recent work on combinatorial surface modifications that aim to control biological responses. Modification and characterization methods for enhanced control over multifunctional material properties are highlighted and discussed. Thereby, this review deepens the understanding and knowledge of biomimetic combinatorial material modification, their challenges but especially their potential.

To determine if the tendon-specific crosslinking gelatin (Col-Tgel) impregnated with growth factors promotes tendon healing at the bone interface and in a tendon window model.

Two different Col-Tgel formulations were first tested in vitro by evaluating cell morphology and tendogenic differentiation. After the optimum formulation was determined, the gel was mixed with either transforming growth factor-β3 (TGF-β3) or growth differentiation factor-7 (GDF-7) growth factor and prepared for injections. Window defects were induced in 12 animals, which were randomized into the following treatments: (1) sham, (2) empty Col-Tgel, (3) Col-Tgel containing TGF-β3, or (4) Col-Tgel containing GDF-7. Based on these results, the sham, empty Col-Tgel, and Col-Tgel containing TGF-β3 were applied to the supraspinatus repair interface. Tendons were analyzed biomechanically and histologically using hematoxylin and eosin and Masson's trichrome staining.

In the window defect model, histologic scores were the best in rats treated with TGF-β3 containing Col-Tgel, followed by the empty Col-Tgel scaffold, and finally the sham control. The GDF-7 Col-Tgel was not further tested because occasional ectopic cartilage and bone formation was found in the prior window defect model. In the supraspinatus repair model, there was no statistical difference (P > .05) in the biomechanical strength among the 3 treatment groups, but load-to-failure ratio improved when TGF-β3 was added to the scaffold, suggesting improved tendon healing.

This pilot study evaluated the performance of an injectable gel tendon graft in a population of retired breeder rats. The results suggest that Col-Tgel containing TGF-β3 may be a useful adjunctive treatment for surgical repair of full-thickness rotator cuff tears. Histologic and biomechanical scores suggest that Col-Tgel containing TGF-β3 promotes tendon healing.

The results of this study suggest that shoulders injected with Col-Tgel may be a useful adjunctive treatment for repair of rotator cuff tears.

Hydrogels represent an attractive material platform for realization of three-dimensional (3D) tissue-engineered constructs, as they have tunable mechanical properties, are compatible with different types of cells, and resemble elements found in natural extracellular matrices. So far, numerous hydrogel-cartilage/bone tissue engineering (TE)-related studies were performed by utilizing a single cell encapsulation approach. Although multicellular spheroid cultures exhibit advantageous properties for cartilage or bone TE, the chondrogenic or osteogenic differentiation potential of stem cell microspheroids within hydrogels has not been investigated much. This study explores, for the first time, how stiffness of gelatin-based hydrogels (having a storage modulus of 538, 3584, or 7263 Pa) affects proliferation and differentiation of microspheroids formed from telomerase-immortalized human adipose-derived stem cells (hASC/hTERT). Confocal microscopy indicates that all tested hydrogels supported cell viability during their 3- to 5-week culture period in the control, chondrogenic, or osteogenic medium. Although in the softer hydrogels cells from neighboring microspheroids started outgrowing and interconnecting within a few days, their protrusion was slower or limited in stiffer hydrogels or those cultured in chondrogenic medium, respectively. High expressions of chondrogenic markers (SOX9, ACAN, COL2A1), detected in all tested hydrogels, proved that the chondrogenic differentiation of hASC/hTERT microspheroids was very successful, especially in the two softer hydrogels, where superior cartilage-specific properties were confirmed by Alcian blue staining. These chondrogenically induced samples also expressed COL10A1, a marker of chondrocyte hypertrophy. Interestingly, the hydrogel itself (with no differentiation medium) showed a slight chondrogenic induction. Regardless of the hydrogel stiffness, in the samples stimulated with osteogenic medium, the expression of selected markers RUNX2, BGLAP, ALPL, and COL1A1 was not conclusive. Nevertheless, the von Kossa staining confirmed the presence of calcium deposits in osteogenically stimulated samples in the two softer hydrogels, suggesting that these also favor osteogenesis. This observation was also confirmed by Alizarin red quantification assay, with which higher amounts of calcium were detected in the osteogenically induced hydrogels than in their controls. The presented data indicate that the encapsulation of adipose-derived stem cell microspheroids in gelatin-based hydrogels show promising potential for future applications in cartilage or bone TE. Impact Statement Osteochondral defects represent one of the leading causes of disability in the world. Although numerous tissue engineering (TE) approaches have shown success in cartilage and bone tissue regeneration, achieving native-like characteristics of these tissues remains challenging. This study demonstrates that in the presence of a corresponding differentiation medium, gelatin-based hydrogels support moderate osteogenic and excellent chondrogenic differentiation of photo-encapsulated human adipose-derived stem cell microspheroids, the extent of which depends on hydrogel stiffness. Because photosensitive hydrogels are a convenient material platform for creating stiffness gradients in three dimensions, the presented microspheroid-hydrogel encapsulation strategy holds promise for future strategies of cartilage or bone TE.

Multiple myeloma is a malignant disease characterized by accumulation of clonal plasma cells in the bone marrow. Uncoupling of bone formation and resorption by myeloma cells leads to osteolytic lesions. These are prone to fracture and represent a possible survival space for myeloma cells under treatment causing disease relapse. Here we report on a novel approach suitable for local treatment of multiple myeloma based on hyaluronic acid (HA) hydrogels mimicking the physical properties of the bone marrow. The HA hydrogels are complexed with heparin to achieve sustained presentation and controlled release of bone morphogenetic protein 6 (BMP-6). Others and we have shown that BMP-6 induces myeloma cell apoptosis and bone formation. Using quartz crystal microbalance and enzyme-linked immunosorbent assay, we measured an initial surface density of 400 ng BMP6/cm², corresponding to two BMP-6 per heparin molecule, with 50% release within two weeks. HA-hydrogels presenting BMP-6 enhanced the phosphorylation of Smad 1/5 while reducing the activity of BMP-6 antagonist sclerostin. These materials induced osteogenic differentiation of mesenchymal stromal cells and decreased the viability of myeloma cell lines and primary myeloma cells. BMP-6 functionalized HA-hydrogels represent a promising material for local treatment of myeloma-induced bone disease and residual myeloma cells within lesions to minimize disease relapse or fractures. STATEMENT OF SIGNIFICANCE: Multiple myeloma is a hematological cancer characterized by the accumulation of clonal plasma cells in the bone marrow and local suppression of bone formation, resulting in osteolytic lesions and fractures. Despite recent advances in systemic treatment of multiple myeloma, it is rare to achieve a targeted suppression of myeloma cells and healing of bone lesions. Here we present hydrogels which mimic the physico-chemical properties of the bone marrow, consisting of hyaluronic acid with crosslinked heparin for the controlled presentation of bioactive BMP-6. The hydrogels decrease the viability of myeloma cell lines and primary myeloma cells and induces osteogenic differentiation of mesenchymal stromal cells. The presentation of BMP-6 in the hyaluronan hydrogels enhances the phosphorylation of Smad1/5 while reducing the activity of the BMP-6 antagonist sclerostin. As such, BMP-6 functionalized hyaluronan hydrogels represent a promising material for the localized eradication of myeloma cells.

Bone tissue engineering has been continuously developing since the concept of "tissue engineering" has been proposed. Biomaterials that are used as the basic material for the fabrication of scaffolds play a vital role in bone tissue engineering. This paper first introduces a strategy for literature search. Then, it describes the structure, mechanical properties and materials of natural bone and the strategies of bone tissue engineering. Particularly, it focuses on the current knowledge about biomaterials used in the fabrication of bone tissue engineering scaffolds, which includes the history, types, properties and applications of biomaterials. The effects of additives such as signaling molecules, stem cells, and functional materials on the performance of the scaffolds are also discussed.

Recently, we found that although high-stiffness matrices stimulated osteogenic differentiation of bone marrow-derived stromal cells (BMSCs), the macrophages (Mφs) in high-stiffness transglutaminase crosslinked gelatins (TG-gels) tended to undergo M1 polarization and hence compromised cell osteogenesis. In this study, we hypothesized that the copresentation of interleukin (IL)-4 and stromal cell-derived factor (SDF)-1α in high-stiffness TG-gels may enhance periodontal regeneration by modulating Mφ polarization and promoting endogenous stem cell recruitment. We found that Mφs were more likely to polarize toward an immunomodulatory M2 state in the presence of IL-4 and hence positively influence the osteogenic differentiation of BMSCs when these cells coexisted in either indirect or direct co-culture systems. In cell migration assays, BMSCs exhibited an enhanced capability to move toward gels containing SDF-1α, and more cells could be recruited into the three-dimensional matrix of TG-gels. When TG-gels containing IL-4 and/or SDF-1α were used to repair periodontal defects, more new bone (MicroCT) was formed in animals that received the dual cytokine-loaded transplants at 4 weeks postsurgery. Mφs were recruited to all the transplanted gels, and after one week, more M1-phenotype cells were found in the groups without IL-4, while the presence of IL-4 was more likely to result in M2 polarization (immunofluorescence staining). When the tissue biopsies were histologically examined, the TG-gels containing both IL-4 and SDF-1α led to a generally satisfactory regeneration with respect to attachment recovery (epithelial and connective tissue) and hybrid tissue regeneration (bone, periodontal ligament and cementum). Our data suggest that the incorporation of IL-4 into high-stiffness TG-gels may promote the M2 polarization of Mφs and that SDF-1α can be applied to guide endogenous cell homing. Overall, building capacity for Mφ modulation and cell recruitment in high-stiffness hydrogels represents a simple and effective strategy that can support high levels of periodontal tissue regeneration. STATEMENT OF SIGNIFICANCE: The development of hydrogel-based regenerative therapies centered on the mobilization and stimulation of native cells for therapeutics opens a window toward realizing periodontal endogenous regeneration. In the present study, the parallel use of immunomodulatory and homing factors in high-stiffness hydrogel materials is shown to induce stem cell homing, modulate cell differentiation and indeed induce regrowth of the periodontium. We found that incorporation of interleukin (IL)-4 in high-stiffness TG-gels coaxed macrophages to polarize into M2 phenotypes, and stromal cell-derived factor (SDF)-1α could be applied to direct endogenous cell homing. Hence, we present for the first time a clinically relevant strategy based on macrophage modulation and host cell recruitment that can support high levels of periodontal tissue regeneration.

For stem cell differentiation, the microenvironment can play an important role, and hydrogels can provide a three-dimensional microenvironment to allow native cell growth in vitro. A challenge is that the stem cell's differentiation can be influenced by the matrix stiffness. We demonstrate a low-toxicity method to create different stiffness matrices, by using a photopolymerizable gelatin methacrylate (GelMA) hydrogel cross-linked by blue light (440 nm). The stiffness and porosity of GelMA hydrogel is easily modified by altering its concentration. We used human bone marrow mesenchymal stem cells (MSCs) as a cell source and cultured the GelMA-encapsulated cells with EGM-2 medium to induce endothelial differentiation. In our GelMA blue light hydrogel system, we found that MSCs can be differentiated into both endothelial-like and osteogenic-like cells. The mRNA expressions of endothelial cell markers CD31, von Willebrand factor, vascular endothelial growth factor receptor-2, and CD34 were significantly increased in softer GelMA hydrogels (7.5% and 10%) compared with stiffer matrices (15% GelMA). On the other hand, the enhancements of osteogenic markers mRNA expressions (Alkaline phosphatase (ALP), Runx2, osteocalcin, and osteopontin) were highest in 10% GelMA. We also found that 10% GelMA hydrogel offered optimal conditions for MSCs to form capillary-like structures. These results suggest that the mechanical properties of the GelMA hydrogel can influence both endothelial and osteogenic differentiation of MSCs and sequent capillary-like formation.

Electrical stimulation (ES) and magnetic stimulation (MS) can promote bone tissue formation in vivo. Loading ES and MS simultaneously would be very beneficial for bone tissue construction in vitro or in vivo. Magnetic-conductive bi-functional scaffolds which are favorable for the transfer of ES and MS, could further facilitate bone cell/tissue growth. Poly(3,4-ethylenedioxythiophene) (PEDOT)/Fe₃O₄/polylactic acid-co-glycolic acid (PLGA) magnetic-conductive bi-functional fibrous scaffolds were prepared through in situ polymerization of EDOT on Fe₃O₄/PLGA fibers. MC3T3-E1 pre-osteoblasts were incubated on the PEDOT/Fe₃O₄/PLGA fibrous scaffolds and were stimulated by electrical, magnetic and electrical-magnetic signals respectively to detect the impact of different stimulation on cell viability. The measured results show that the scaffolds possess good conductivity and superparamagnetic responsiveness. Furthermore, both electrical and magnetic stimulation promoted cell proliferation and magnetic stimulation could induce cell alignment arrangement. Meanwhile, under electrical-magnetic double stimulation, cell viability was much higher than for cells under single electrical or magnetic stimulation. The growth promoting effects of PEDOT/Fe₃O₄/PLGA fibrous scaffolds under electrical-magnetic double stimulation has great practical potential for bone tissue engineering.